Method for production of neuroblasts

ABSTRACT

A method for producing a neuroblast and a cellular composition comprising an enriched population of neuroblast cells is provided. Also disclosed are methods for identifying compositions which affect neuroblasts and for treating a subject with a neuronal disorder, and a culture system for the production and maintenance of neuroblasts.

This application is a Continuation of U.S. application Ser. No.10/622,206, filed Jul. 18, 2003, now abandoned, which is a Continuationof U.S. application Ser. No. 09/915,229, filed Jul. 24, 2001, now U.S.Pat. No. 6,599,695, which is a Continuation of U.S. application Ser. No.08/884,427, filed Jun. 27, 1997, now U.S. Pat. No. 6,265,175, which is aContinuation of U.S. application Ser. No. 08/445,075, filed May 19,1995, now abandoned, which is a Divisional of U.S. application Ser. No.08/147,843, filed Nov. 3, 1993, now U.S. Pat. No. 5,766,948, which is aContinuation-in-part of U.S. application Ser. No. 08/001,543, filed Jan.6, 1993, now abandoned. The contents of each of these prior applicationsare incorporated by reference herein in their entirety for any purpose.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to cell populations derived fromneurons, which are denoted neuroblasts, methods for the production andlong-term in vitro culture of these cell populations, and the use ofneuroblasts in the treatment of various neuronal disorders as well asthe identification of compositions which affect neuroblasts.

2. Description of Related Art

Only a few neuronal cell types have been reported to divide in the adultbrain and adult neurons do not survive well in vitro. To date, even withall of the recent advances in neurobiology, genetics, immunology andmolecular biology, no reliable procedure exists to establish cell linesfrom the central nervous system (CNS) and neuronal tissues in theabsence of immortalization. The generation of clonal cell lines fromdifferent regions of the brain is important and will greatly facilitatethe discovery of new neurotrophic factors and their receptors, andenhance the understanding of their function.

The central nervous system contains two major classes of cells known asneurons and glial cells. Glial cells include astrocytes,oligodendrocytes and microgliA. There are hundreds of different types ofneurons and many different neurotrophic factors which influence theirgrowth and differentiation. Depending on the type of neuron and theregion of the brain in which the neuron resides, a differentneurotrophic factor or specific combination of factors affect thesurvival, proliferation and differentiation of the neuron. Each type ofneuron responds to different combinations of neurotransmitters,neurotrophic factors, and other molecules in its environment.

To date, neuropharmacologicai studies in the CNS have been delayed bythe lack of cell systems needed to investigate potentially usefulneuroactive compounds. In live animals, the complexity of the brainmakes it difficult to effectively measure which cellular receptors arebeing targeted by these compounds. Additionally, the expense involved inlive animal research and the current controversies stemming from animalrights movements have made in vivo animal studies less acceptable forinitial research. Primary cells from neuronal tissue are often used forCNS studies, however, long-term culture of primary neurons has not beenachieved. Also, only a few attempts to achieve not only long termculture, but actual proliferation of neuronal cells have been reported.In fact, the proliferation of neuronal cells has proven so elusive thatit has become ingrained in the scientific community that neuronal cellsdo not proliferate in vitro. As a consequence, fresh dissections must beperformed for each study in order to obtain the necessary neuronal celltypes, resulting in costly research with increased variability in-theexperimental results.

While some neuronal tumorogenic cells exist they are few in number andare not well characterizeD. In general, these tumor cell lines do notmimic the biology of the primary neurons from which they were originallyestablished and, as a result, are not suitable for drug discoveryscreening programs. In vitro primary cultures that would be morephenotypically representative of primary cells and that could generatecontinuous cultures of specific neuronal cell lines capable ofproliferation would be invaluable for neurobiological studies and CNSdrug discovery efforts, as well as therapy.

It has become increasingly apparent that more defined conditions andfurther refinements in culture methodology are necessary to produceneuronal cell lines which would enhance the yield of information from invitro studies of the nervous system. Recognition of cell type anddevelopmental stage-specific requirements for maintaining neural cellsin culture as well as the development of a broader range of cultureconditions are required. However, in order to achieve these goals it iscritical to develop optimal culture methods which mimic in vivoconditions which are devoid of the biological fluids used inconventional culture techniques.

Recently, several researchers have isolated and immortalized progenitorcells from various regions of the brain and different stages ofdevelopment. Olfactory and cerebellum cells have been immortalized usingthe viral myc (v-myc) oncogene to generate-cell lines with neuronal andglial phenotypes (Ryder, et al., J. Neurobiology, 21:356, 1990). Similarstudies by Snyder, et al. (Cell, 68:33, 1992) resulted in multipotentneuronal cell lines which were engrafted into the rat cerebellum to formneurons and glial cells. In other studies, murine neuroepithelial cellswere immortalized with a retrovirus vector containing c-myc and werecultured with growth factors to form differentiated cell types similarto astrocytes and neurons (Barlett et al., Proc. Natl Acad. Sci. USA,85:3255, 1988).

Epidermal growth factor (EGF) has been used to induce the in vitroproliferation of a small number of cells isolated from the striatum ofthe adult mouse brain (Reynolds and Weiss, Science, 255:1707 1992).Clusters of these cells had antigenic properties of neuroepithelial stemcells and under appropriate conditions, these cells could be induced todifferentiate into astrocytes and neurons with phenotypes characteristicof the adult striatum in vivo. However, it should be noted that thesedifferentiated neurons were not cultured for lengthy periods of time norwas there any evidence that these cells could be frozen and then thawedand recultured.

Cattaneo and McKay (Nature, 347:762, 1990) performed experiments usingrat striatum to determine the effect of nerve growth factor (NGF) onproliferation of neuronal precursor cells. The cells were dissected fromrat embryonic striatum and exposed to both NGF and basic fibroblastgrowth factor (bFGF, also known as FGF2). These cells were cultured onlynine days in vitro, at which time they had differentiated into neuronsas determined by assay with neuron-specific markers.

Neuronal precursor cells from the cerebral hemispheres of 13-day old ratembryos have been cultured for up to 8 days in the presence of bFGF at 5ng/ml (Gensberger, et al., FEBS Lett. 217:1, 1987). At thisconcentration, bFGF stimulated only short-term proliferation.Proliferation and differentiation of primary neurons from both fetal andadult striatum in response to a combination of NGF and bFGF or only EGFhave also been reported (Catteneo, et al., supra, Reynolds and Weiss,supra).

In view of the foregoing, there is a need for a long-term in vitroculture system which would allow large scale production and maintenanceof a neuronal cell population which will proliferate and can be passagedand subcultured over time. Such homogenous in vitro neuronal cultureswill prove invaluable in studying cell populations, the interactionsbetween these cells and the effects of various neuroactive compositionson these cells.

SUMMARY OF THE INVENTION

Recognizing the importance of a system for producing and maintainingneuronal cells in vitro, the inventors developed a method and a culturesystem for producing continuous fetal and adult neuronal cell lines. Thedevelopment of primary neuronal cultures maintained as cell lines, knownas neuroblasts, using neurotrophic factors in the absence of oncogenicimmortalization, now permits investigation of fundamental questionsregarding the biochemical and cellular properties of these cells and thedynamics of interaction between their cellular and chemical environment.

The neuroblasts of the invention can advantageously be used to stablyincorporate genetic sequences encoding various receptors, ligands andneurotransmitters, for example, for use in the treatment of subjectswith neuronal disorders and for identifying compositions which interactwith these molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show BrdU staining and NeuroTag™ binding of primary neuronsin culture. FIG. 1A. Primary neurons were labeled with BrdU for 1 day;and FIG. 1B for 4 days. FIG. 1C. The neuronal nature of primary cellswas determined by binding with tetanus toxin (NeuroTag™). Cell bodiesand processes of all cells in culture were stained. Calibration bar=20μm.

FIGS. 2A-2F illustrates photomicrographs showing the morphologicalchanges that occur during the culture and passaging of primary neurons.FIG. 2A. Primary cell culture after 4 days of plating in N2+bFGF. FIG.2B. Primary cells 4 days in culture after passage (passage 3). Cellswere larger and interconnected by processes that also increased in size.Small proliferating cells were visible in the culture. FIG. 2C. Cellspassaged (passage 3) and kept in culture for ˜14 days in the presence ofbFGF. Negative magnification 33×. FIG. 1D. Cells immunostained withanti-NF (200 KD) antibody, negative magnification 33×. FIG. 1E. Cellsiminunostained with anti-NSE antibody at negative magnification 33×.FIG. 1F. Cells immunostained with anti-GFAP antibody at negativemagnification 66×.

FIGS. 3A-3D show transmission electron micrographs of primary neurons inculture. FIG. 3A. A pyramidal-shaped primary hippocampal neuron showingboth the soma and processes, including a major apical process (arrow)and a finer caliber process (arrowhead). Bar=10 μm. FIG. 3B. Enlargedview of the neuronal soma shown in FIG. 3A. Bar=1 μm. FIG. 3C. A portionof the major apical process of the neuron shown in FIG. 3A. Bar=1 μm.FIG. 3D. Contact between two neuritic processes. Bar=0.1 μm.

FIGS. 4A-4D show scanning electron micrographs of primary neurons inculture. FIG. 4A. Overview of primary hippocampal neurons in cultureincluding well-differentiated pyramidal somata (arrow) with largeprocesses containing multiple levels of branching andless-differentiated, rounded neurons with large, extended processes(arrowheads). Bar=50 μm. FIG. 4B. A major apical dendrite emerging froma well-differentiated pyramidal neuron showing a smooth, regular caliberprocess just proximal to the first (major) bifurcation with severalsmaller processes, possibly axons emerging from it. The PORN/laminincoating the vessel surface can be seen as a porous carpeting which isabsent in some patches. Bar=2 μm. FIG. 4C. A well-differentiated neuron(in the middle of the field) possessing a large pyramidal soma (compareto FIG. 3A) and a large apical dendrite (arrowheads) contacted by anumber of processes from other neurons. Other less-differentiatedneurons which are fixed in the process of dividing were also present(arrows). Bar=20 μm. FIG. 4D. Enlarged view of the dividing neuron inthe upper field of view in FIG. 4C. Bar=10 μm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an in vitro method for producing anisolated neuronal cell population. These cells, termed neuroblasts canbe produced by utilizing methodology which comprises culturing aneuronal cell in a serum-free media supplemented with at least onetrophic factor using a vessel which allows attachment of the cell. Thismethod allows the generation of continuous, neuronal cell cultures fromdifferent regions of the brain, from both fetal and adult tissue, whichare capable of proliferation.

The invention also provides a method of identifying compositions whichaffect a neuroblast, such as by inhibiting or stimulating the neuroblastproliferation. A culture system useful for the production andmaintenance of a neuroblast comprising a serum-free basal mediacontaining at least one trophic factor and a vessel which allowsattachment of the neuroblast is also provided. An enriched population ofneuroblast cells produced by the method of the invention is alsoprovided-and can be further utilized for the treatment of a subject witha neuronal cell disorder or alternatively, to screen compositions whichaffect the neuroblast.

As used herein, the term “neuroblast” refers to a non-glial cell ofneuronal lineage which has been perpetualized. Neuronal“perpetualization” refers to the procedure whereby a non-glial cell ofthe neuronal lineage is treated with growth factors such that it iscapable of indefinite maintenance, growth and proliferation in vitro.Typically, a primary culture, one in which the tissue is removed from ananimal, is placed in a culture vessel in appropriate fluid medium, andhas a finite lifetime. In contrast, continuous cell lines proliferateand thus can be subcultured, i.e., passaged repeatedly into new culturevessels. Continuous cell lines can also be stored for long periods oftime in a frozen state in the vapor phase of liquid nitrogen when acryopreservative is present, e.g., 10% dimethylsulfoxide or glycerol.The neuroblast of the invention can be maintained in long-term cultureas a cell line closely resembling primary cultures, but without resortto oncogenic immortalization. Rather, “perpetualization” establishes acontinuous culture from a primary neuronal cell by utilizing a specificgrowth factor or combination of growth factors. This perpetualizationtechnique is novel in that no gene transfer or genetic manipulation isrequired and, as a consequence, the cells more closely resemble primarycultures.

There are hundreds of different types of neurons, each with distinctproperties. Each type of neuron produces and responds to differentcombinations of neurotransmitters and neurotrophic factors. Neurons donot divide in the adult brain, nor do they generally survive long invitro. The method of the invention provides for the isolation and growthof perpetualized neurons, or neuroblasts, in vitro, from virtually anyregion of the brain and spinal cord. Either embryonic or adult neuronscan be utilized for the development of neuroblast cell lines. Theneuronal cell of the invention, which is-utilized for production of aneuroblast, can be derived from any fetal or adult neural tissue,including tissue from the hippocampus, cerebellum, spinal cord, cortex(e.g., motor or somatosensory cortex), striatum, basal forebrain(cholenergic neurons), ventral mesencephalon (cells of the substantianigra), and-the locus ceruleus (neuroadrenaline cells of the centralnervous system).

The liquid media for production of a neuroblast of the invention issupplemented with at least one trophic factor to support the growth andproliferation of a neuroblast Trophic factors are molecules which areinvolved in the development and survival of neurons. They are oftensynthesized in the brain, have specific receptors, and influence thesurvival and function of a subset of neurons. Examples of such factorsinclude nerve growth factor (NGF), brain-derived neurotrophic factor(BDNF), neurotrophin-3, -4, and -5 (NTF-3, -4, -5), ciliary neurotrophicfactor (CNTF), basic fibroblast growth factor (bFGF), acidic fibroblastgrowth factor (aFGF), platelet derived growth factor (PDGF), epidermalgrowth factor (EGF), insulin-like growth factor-I and -II (IGF-I, -II),transforming growth factor (TGF) and lymphocyte infiltratingfactor/cholinergic differentiating factor (LIF/CDF). The specificity andselectivity of a trophic factor are determined by its receptor.Preferably, the trophic factor utilized in the invention is aneurotrophic factor. Preferably, the neurotrophic factor added to thebasal media for production of a neuroblast according to the method ofthe invention is bFGF. The neurotrophic factor which allows growth andproliferation of the neuroblast in vitro will depend on the tissueorigin of the neuroblast. However, for most neuronal cells, bFGF will bethe preferred neurotrophic factor.

The vessel utilized for production of a neuroblast must provide asurface which allows attachment of the neuronal cell. Such vessels arealso preferred once the isolated neuroblast culture has been produced.The surface used to enhance attachment-of the neuronal cell can be theactual inner layer of the vessel or more indirectly, the surface of asupplemental insert or membrane which resides within the vessel.Attachment may be accomplished by any means which allows the cell togrow as a monolayer on a vessel. Attachment enhancing surfaces can beproduced directly, such as by advantageous selecting of appropriateplastic polymers for the vessel or, indirectly, as by treating thesurface in the vessel by a secondary chemical treatment Therefore,“attachment” refers to the ability of a cell to adhere to a surface in atissue culture vessel, wherein the attachment promoting surface is indirect contact with neuronal cells, which otherwise would grow in athree-dimensional cellular aggregate in suspension. Attachment oradherence, of a neuronal cell to the vessel surface allows it to beperpetualized.

In addition to interactions with soluble factors, most cells in vivo,including neuronal cells, are in contact with an extracellular matrix, acomplex arrangement of interactive protein and polysaccharide moleculeswhich are secreted locally and assemble into an intricate network in thespaces between cells. Therefore, the addition of an extracellular matrixprotein to the surface of the culture vessel forms an insoluble matrixwhich allows neuronal cells in culture to adhere in a manner whichclosely corresponds to the in vivo extracellular matrix The neuroblastof the invention can be preferably produced by coating the surface of avessel, such as a tissue culture dish or flask, with a polybasic aminoacid composition to allow initial attachment. Such compositions are wellknown in the art and include polyomithine and pofylysine. Mostpreferably, the polybasic amino acid of the invention is polyomithine.Additionally, the surface of the vessel may be coated with a knownextracellular matrix protein composition to enhance the neuroblast'sability to grow and form processes on the substrate. Such compositionsinclude laminin, collagen and fibronectin. Other extracellular matrixproteins that can be used in conjunction with a polybasic amino acidwill be apparent to one of skill in the art. Additionally, for theproduction of adult neuroblasts, it is preferable to initially culturethe cells in the presence of serum.

The neuroblast of the invention is useful as a screening tool forneuropharmacological compounds which affect a biological function of theneuroblast Thus, in another embodiment, the invention provides a methodfor identifying a composition which affects a neuroblast comprisingincubating the components, which include the composition to be testedand the neuroblast, under conditions sufficient to allow the componentsto interact, then subsequently measuring the effect the composition onthe neuroblast. The observed effect on the neuroblast may be eitherinhibitory or stimulatory. For example, a neuroactive compound whichmimics a neurotransmitter or binds to a receptor and exhibits either anantagonistic or agonist effect thereby inhibiting or stimulating abiological response in the neuroblast, can be identified using themethod of the invention. The occurrence of a biological response can bemonitored using standard techniques known to those skilled in the art.For example, inhibition or stimulation of a biological response may beidentified by the level of expression of certain genes in the neuroblastSuch genes may include early response genes such as fos, myc or jun(Greenberg, M. and Ziff, E. Nature, 311:433, 1984; eds. Burck, et al.,in Oncogenes, 1988, Springer-Verlag, New York.). Other genes, includingthose which encode cell surface markers can also be used as indicatorsof the effects neuropharmacological compounds on the neuroblasts of theinvention. Methods for measurement of such effects include Northern blotanalysis of RNA (transcription), SDS-PAGE analysis of protein(translation), [³H]-thymidine uptake (DNA synthesis) and antibodyreactivity (both intracellular and extracellular). Other commonly usedmethods will be apparent to those of skill in the art.]

Neuroactive drugs which act similarly to those already known to affectneuronal cells can thus be identified. For example, new drugs thatalleviate anxiety, analogously to Valium, which augment or stimulate theaction of the important inhibitory transmitter gamma-aminobutyric add(GABA), can be identified. Antidepressants, such as Prozac, enhance theaction of serotonin, an indoleamine with a wide variety of functions.Other drugs can be readily identified using the neuroblasts according tothe method of the invention. Other examples include psychoactivecompounds. For example, cocaine facilitates the action of dopamine,whereas certain antipsychotics antagonize or inhibit this catecholamine.Another example is nicotine which activates the acetylcholine receptorswhich are distributed throughout the cerebral cortex. Therefore, byusing neuroblasts derived from neuronal cells from the appropriateregions of the brain, drugs and trophic factors which bind variousreceptors and would produce similar effects on neuronal cells can beidentified using the method of the invention.

As described above, perpetualization of a neuronal cell can beaccomplished without the use of oncogenic intervention. However, ifdesired the neuroblast of the invention may be immortalized to maintainthe cell at a defined developmental stage. The present techniques forimmortalization typically involve the transfection of an oncogene to theceil, therefore, immortalization of a neuroblast can be achieved byintroduction of at least one oncogene to the neuroblast. Transfection ofthe oncogene can be accomplished by several conventional methods wellknown to those skilled in the art, including using recombinantretroviruses, chemical, or physical methods. Recombinant retrovirustransfer is the preferred method of the invention for immortalization ofneuroblasts.

The host neuroblast can be immortalized with a particular oncogene bysuch methods of transfection as calcium phosphate co-precipitation,conventional mechanical procedures such as microinjection, insertion ofa plasmid encased in liposomes, or by use of viral vectors. For example,one method is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transform theneuroblast (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory,Gluzman ed., 1982).

Various viral vectors which can be utilized for immortalization astaught herein include adenovirus, herpes virus, vaccinia, andpreferably, an RNA virus such as a retrovirus. Preferably, theretroviral vector is a derivative of a murine or avian retrovirus.Examples of retroviral vectors in which a single foreign gene can beinserted include, but are not limited to: Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additionalretroviral vectors can incorporate multiple genes. All of these vectorscan transfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus (gag, env, andpol genes) under the control of regulatory sequences within the longterminal repeat (LTR). These plasmids are missing a nucleotide sequencewhich enables the packaging mechanism to recognize an RNA transcript forencapsidation. Helper cell lines which have deletions of the packagingsignal include, but are not limited to ψ2, PA317, PA12, CRIP and CRE,for example. These cell lines produce empty virions, since no genome ispackaged, if a retroviral vector is introduced into such cells in whichthe packaging signal is intact, but the structural genes are replaced byother genes of interest, the vector can be packaged and vector virionproduced. The vector virions produced by this method can then be used toinfect a tissue cell line, such as NIH 3T3 cells, to produce largequantities of chimeric retroviral virions.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate or lipofectiontransection. These cells are then transfected with the vector plasmidcontaining the genes of interest. The resulting cells release theretroviral vector into the culture medium.

Herpes virus-based vectors may also be used to transfer genes into aneuroblast Since herpes viruses are capable of establishing a latentinfection and an apparently non-pathogenic relationship with some neuralcells, such vector based on HSV-1, for example, may be used. Similarly,it should be possible to take advantage other human and animal virusesthat infect cells of the CNS efficiently, such as rabies virus, measles,and other paramyxoviruses and even the human immunodeficiency retrovirus(HIV), to develop useful delivery and expression vectors.

When a recombinant retrovirus is engineered to contain an immortalizingoncogene, the oncogene can be any one of those known to immortalize. Forexample, such commonly used immortalizing genes include genes of the mycfamily (both c-myc and v-myc) (Barlett, et al., Proc. Natl. Acad. Sci.USA 85:3255, 1988), adenovirus genes (E1a 12s and E1a 13s) (Ruley, etal., Nature 304:602, 1983), the polyoma large T antigen and SV40 large Tantigen (Frederiksen, et al., Neuron 1:439, 1988). Preferably, theoncogene used to immortalize the neuroblast of the invention is v-myc.Other genes, for example other nuclear oncogenes, that immortalize acell but may require a second gene for complete transformation, will beknown to those of skill in the art.

The same transfection methods described above for immortalization of aneuroblast can be utilized to transfer other exogenous genes to theneuroblast of the invention. An “exogenous gene” refers to geneticmaterial from outside the neuroblast which is introduced into theneuroblast. An example of a desirable exogenous gene which would beuseful for the method of identifying neuropharmacological compounds is agene for a receptor molecule. For example, such neuronal receptorsinclude the receptor which binds dopamine, GABA, adrenaline,noradrenaline, serotonin, glutarnate, acetylcholine and various otherneuropeptides. Transfer and expression of a particular receptor in aneuroblast of specific neural origin, would allow identification ofneuroactive drugs and trophic factors which may be useful for thetreatment of diseases involving that neuroblast cell type and thatreceptor. For example, a neuroactive compound which mimics aneurotransmitter and binds to a receptor and exhibits either anantagonistic or agonist effect, thereby inhibiting or stimulating aresponse in the neuroblast, can be identified using the method of theinvention.

In another embodiment, the invention provides a culture system usefulfor the production and maintenance of a neuroblast comprising aserum-free basal media containing at least one trophic factor and avessel having a surface which allows attachment of the neuroblast. Theculture system can be utilized to produce a neuroblast from any tissueof neural origin as described above. The “serum-free basal media” of theinvention refers to a solution which allows the production andmaintenance of a neuroblast. The basal media is preferably a commonlyused liquid tissue culture media, however, it is free of serum andsupplemented with various defined components which allow the neuroblastto proliferate. Basal media useful in the culture system of theinvention is any tissue culture media well known in the art, such asDulbecco's minimal essential media, which contains appropriate aminoacids, vitamins, inorganic salts, a buffering agent, and an energysource. Purified molecules, which include hormones, growth factors,transport proteins, trace elements, vitamins, and substratum-modifyingfactors are added to the basal media to replace biological fluids. Forexample, progesterone, sodium selenite, putrescine, insulin andtransferrin are typically added to the basal media to enhance neuroblastgrowth and proliferation. For the culture system of the.-invention, onlytwo of the defined supplements are necessary to sustain growth ofneurons alone (transferrin and insulin), whereas the combination of thefive supplements above have a highly synergistic growth-stimulatingeffect Deletion of any single supplement results in markedly diminishedgrowth of the neuroblast. An example of a preferred prototype mediumwhich contains these elements is N2 medium (Bottenstein and Sato, etal., Proc. Natl. Acad. Sci. USA, 76:514, 1979). The optimalconcentration of the supplements are as follows: 5 μg/ml insulin, 100μg/ml transferrin, 20 nM progesterone, 100 μM putrescine, and 30 nMselenium (as Na₂SeO₃).

The basal media of the culture system further contains at least onetrophic factor for the production and maintenance of a neuroblast Mostpreferably, neurotrophic factors are utilized and specifically bFGF.bFGF is utilized in the basal media at a concentration from about 1ng/ml to about 100 ng/ml, more specifically from about 5 ng/ml to about70 ng/ml, and most preferably from about 15 ng/ml to about 60 ng/ml.Neural cultures are generally maintained at pH 7.2-7.6. A higherrequirement for glucose is also necessary for neural as opposed tonon-neural cells. Therefore, the basal media of the invention contains aconcentration of from about 0.01% to about 1.0% glucose and preferablyfrom about 0.1% to about 0.6% glucose.

The invention also provides a cellular composition comprising anenriched population of neuroblast cells. The composition preferablycontains a majority of or at least about 90% neuroblasts. The neuroblastcells are derived from any CNS neural tissue such as from any region ofthe brain, as described above, or from the spinal cord. The neuroblastmay be further immortalized with an oncogene, or it may contain anexogenous gene encoding a receptor or a ligand for a receptor.

The present invention also provides a method of treating a subject witha neuronal cell disorder which comprises administering to the subject atherapeutically effective amount of the neuroblast of the invention.“Therapeutically effective” as used herein, refers to that amount ofneuroblast that is of sufficient quantity to ameliorate the cause of theneuronal disorder. “Ameliorate” refers to a lessening of the detrimentaleffect of the neuronal disorder in the patient receiving the therapy.The subject of the invention is preferably a human, however, it can beenvisioned that any animal with a neuronal disorder can be treated withthe neuroblast of the invention. Preferably, the neuroblast is derivedfrom neuronal tissue of the same species as the species of the subjectreceiving therapy.

The method of treating a subject with a neuronal disorder entailsintracerebral grafting of neuroblasts to the region of the CNS havingthe disorder. Where necessary, the neuroblast can be geneticallyengineered to contain an exogenous gene. The disorder may be from eitherdisease or trauma (injury). Neuroblast transplantation, or “grafting”involves transplantation of cells into the central nervous system orinto the ventricular cavities or subdurally onto the surface of a hostbrain. Such methods for grafting will be known to those skilled in theart and are described in Neural Grafting in the Mammalian CNS, Bjorklundand Stenevi, eds., (1985), incorporated by reference herein. Proceduresinclude intraparenchymal transplantation, (i.e., within the host brain)achieved by injection or deposition of tissue within the host brain soas to be apposed to the brain parenchyma at the time of transplantation.

Administration of the neuroblasts of the invention into selected regionsof the recipient subject's brain may be made by drilling a hole andpiercing the dura to permit, the needle of a microsyringe to beinserted. The neuroblasts can alternatively be injected intrathecallyinto the spinal cord region. The neuroblast preparation of the inventionpermits grafting of neuroblasts to any predetermined site in the brainor spinal cord, and allows multiple grafting simultaneously in severaldifferent sites using the same cell suspension and permits mixtures ofcells from different anatomical regions: The present invention providesa method for transplanting various neural tissues, by providingpreviously unavailable proliferating neuroblasts and a culture systemfor production of these neuroblasts in order to grow a sufficient numberof cells for in vitro gene transfer followed by in vivo implantation.

The neuroblast used for treatment of a neuronal disorder may optionallycontain an exogenous gene, for example, an oncogene, a gene whichencodes a receptor, or a gene which encodes a ligand. Such receptorsinclude receptors which respond to dopamine, GABA. adrenaline,noradrenaline, serotonin, glutamate, acetylcholine and otherneuropeptides, as described above. Examples of ligands which may providea therapeutic effect in a neuronal disorder include dopamine,adrenaline, noradrenaline, acetylcholine, gamma-aminobutyric acid andserotonin. The diffusion and uptake of a required ligand after secretionby a donor neuroblast would be beneficial in a disorder where thesubject's neural cell is defective in the production of such a geneproduct. A neuroblast genetically modified to secrete a neurotrophicfactor, such as nerve growth factor, (NGF), might be used to preventdegeneration of cholinergic neurons that might otherwise die withouttreatment. Alternatively, neuroblasts to be grafted into a subject witha disorder of the basal ganglia, such as Parkinson's disease, can bemodified to contain an exogenous gene encoding L-DOPA, the precursor todopamine. Parkinson's disease is characterized by a loss of dopamineneurons in the substantia-nigra of the midbrain, which have the basalganglia as their major target organ. Alternatively, neuroblasts derivedfrom substantia-nigra neuronal cells which produce dopamine could beintroduced into a Parkinson's patient brain to provide cells which“naturally” produce dopamine.

Other neuronal disorders that can be treated similarly by the method ofthe invention include Alzheimer's disease, Huntington's disease,neuronal damage due to stroke, and damage in the spinal cord.Alzheimer's disease is characterized by degeneration of the cholinergicneurons of the basal forebrain. The neurotransmitter for these neuronsis acetylcholine, which is necessary for their survival. Engraftment ofcholinergic neuroblasts, or neuroblasts containing an exogenous gene fora factor which would promote survival of these neurons can beaccomplished by the method of the invention, as described. Following astroke, there is selective loss of cells in the CA1 of the hippocampusas well as cortical cell loss which may underlie cognitive function andmemory loss in these patients. Once identified, molecules responsiblefor CA1 cell death can be inhibited by the methods of this invention.For example, antisense sequences, or a gene encoding an antagonist canbe transferred to a neuroblast and implanted into the hippocampal regionof the brain.

The method of treating a subject with a neuronal disorder alsocontemplates the grafting of neuroblasts in combination with othertherapeutic procedures useful in the treatment of disorders of the CNS.For example, the neuroblasts can be co-administered with agents such asgrowth factors, gangliosides, antibiotics, neurotransmitters,neurohormones, toxins, neurite promoting molecules and antimetabolitesand precursors of these molecules such as the precursor of dopamine,L-DOPA.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLES

The following examples show neuronal proliferation of hippocampal,spinal cord, substania nigra, basal forebrain, and other neuronal tissuecells from fetal rats cultured over 5 months with bFGF. In addition,adult hippocampus was cultured with bFGF in a defined media for morethan 7 months. The examples also provide methodology for the generation,differentiation and long term culture of numerous cell types from fetaland adult neuronal tissue and describe the morphological,immunocytochemical, ultrastructural and molecular characteristics ofproliferating non-neuronal and neuronal cell types in the adult bFGFtreated cultures.

Proliferating cells that incorporated bromodeoxyuridine wereimmunopositive for neuron-specific enolase. Cells with polarizedmorphologies typical of well-differentiated neurons were immunopositivefor the high molecular weight subunit of neurofilament protein (NFh),characteristic of mature neurons, the middle and low subunits ofneurofilament protein and microtubule-associated protein 2 (MAP-2).Cells from adult mammalian hippocampus were capable of proliferation aswell as long-term neurogenesis and neuronal differentiation in vitro.These cells may be a source of replacement cells in neuronal grafting.Further, the induction of proliferation and differentiation of thesecells in vivo would be useful for replacement or augmentation ofneuronal loss or degeneration.

Example 1 Materials and Methods

Materials: DMEM:F12 medium, N2 supplement and laminin were obtained fromGibco/BRL (Bethesda, Md.); polyornithine (PORN) was obtained from Sigma(St. Louis, Mo.). Recombinant bFGF was from Syntex/Synergen Consortium(Boulder, Colo.). Bovine bFGF was purchased from R&D, Minneapolis, Minn.NeuroTag™ green was obtained from Boehringer Mannheim, Indianapolis,Ind. Cell proliferation kit containing bromodeoxyuridine (BrdU),anti-BrdU antibody and streptavidin/Texas Red was purchased fromAmersham, Arlington Heights, Ind. The antibodies used to determine thephenotypes of cells in culture were obtained from the following sourcesand used at the indicated dilutions: polyclonal rabbitanti-neurofilament 200 (NF) (1:500; Chemicon International, Temecula,Calif.), monoclonal anti-neuron specific enolase (NSE) (1:50; DAKO,Carpenteria, Calif.), monoclonal anti-glia fibrillary acidic protein(GFAP) (1:500-1:10,000; Amersham, Arlington Heights, III), monoclonalanti-vimentin (1:800; Boehringer Mannheim, Indianapolis, Ind.),monoclonal anti-OX-42 (1:5000; Serotec, Indianapolis, Ind.), polyclonalanti-galactocerebroside (Gal C) (1:5000; Advanced ImmunochemicalServices, Long Beach, Calif.), monoclonal anti-microtubule associatedprotein (MAP 2) (1:500; Sigma Immunochemicals, St. Louis, Mo.),polyclonal anti-fibronectin (1:2000; Telios, La Jolla, Calif.).Polyclonal nestin antibody (1:15,000) was from Dr. R. McKay, MIT,Cambridge, Mass., and monoclonal high affinity bFGF receptor antibody(1:20) was from Dr. A. Baird, Whittier Institute, La Jolla, Calif.Polyclonal anti-GFAP (1:2000) was from Dr. L. F. Eng, StanfordUniversity, Palo Alto, Calif.

Cell Culture. The brains of Fisher 344 rats (E16) were dissected, themeninges were removed and the hippocampi were isolated. Hippocampi weretransferred to a 15 ml tissue culture tube and the volume was adjustedto 1-2 ml with phosphate-buffered saline (pH 7.4) supplemented with 0.6%glucose (PBS-G). Hippocampi were mechanically dissociated by triturationwith a pasteur pipet (˜20×) followed by trituration with a pasteur pipetfire-polished to significantly reduce the pipet bore (˜20×). The cellsuspension was pelleted by centrifugation at 1000 rpm for 5 minutes atroom temperature. Cells were taken up in ˜20 ml N2 medium (1:1 mixtureof DMEM:F12 containing 20 nM progesterone, 30 nM sodium selenite, 100 μMputrescine, 3.9 mM glutamine, 5 μM/ml insulin, 100 μg/ml transferrin)and the cell number was quantified with a hemocytometer. Tissue cultureplates were coated with polyomithine (PORN; 10 μg/ml) followed bylaminin (10 μg/ml). Approximately 0.5-1.0×10⁶ cells/well were plated onPORN/laminin-coated 6 well plates in N2 medium containing 20 ng/ml bFGF(N2 -bFGF) and cultured at 37° C. in 5% CO₂. Medium was changed every3-4 days with fresh N2+bFGF. For passaging, cells were trypsinized (ATVtrypsin, Irvine Scientific, Santa Ana, Calif.) and then taken up inN2+bFGF. Cells were pelleted by centrifugation and supernatantcontaining trypsin was removed. Cells were resuspended in 10 ml N2+bFGFand plated. Cells could be frozen in liquid nitrogen in N2+bFGF+10%dimethylsulfoxide (DMSO). For culturing, cells were thawed quickly at37° C., added to 10 ml N2+bFGF, centrifuged to remove DMSO, resuspendedin fresh N2+bFGF and plated as described before.

BrdU Incorporation Experiments. Primary neurons (passage 5) were grownfor 3 days, whereupon the media was changed. On the following day cellswere incubated with BrdU for either 1 day or 4 days. Cells were fixed,washed and then treated with a monoclonal antibody against BrdU for 1hour. After washing, cells were reacted with biotinylated anti-mouseantibody (Vector Laboratories, Burlingame, Calif.) followed bystreptavidin/Texas Red complex. Stained cultures were examined with aBioRad MRC600 confocal scanning laser microscope equipped with akrypton-argon laser using the YHS filter set (568 EX, 585 LP). Confocalfluorescent and Nomarski transmitted collected images were transferredto an Apple Macintosh Quadra 700, merged using Adobe Photoshop 2.01, andprinted out on a GCC film recorder.

Neurotag™ Binding. Primary neurons (passage 5) grown in culture for 6days were incubated with 10 μg/ml recombinant tetanus toxin C fragmentconjugated to fluorescein isothiocyanate (NeuroTag™) in N2+bFGF andbovine serum albumin (0.1 mg/ml) for 2 hours. After washing the cellswere examined in a BioRad confocal microscope as described for BrdUstained cells except using the BHS filter (488 EX, 515 LP).

Immunohistochemistry. Cells were passaged (passage 3; 4 days in cultureafter plating), grown in a 24-well plate, fixed in 4% paraformaldehydein PBS, and then permeabilized with 0.25% Triton X-100 in Tris bufferedsaline. Cells were incubated overnight at 4° C. with polyclonal ormonoclonal antibodies in the presence of 1% normal horse serum (formonoclonal antibody) or 10% normal goat serum (for polyclonal antibody).After washing, cells were incubated with biotin conjugated goatanti-rabbit IgG or horse anti-mouse IgG antibodies (Vector Laboratories,Burlingame, Calif.) for 1 hour at room temperature, followed byincubation for 1 hour at room temperature with a pre-formed mixture ofavidin-biotinylated horseradish peroxidase complex (Vectastain Elite ABCkit). The reaction products were visualized with diaminobenzidine (DAB)histochemistry.

Transmission Electron Microscopy (TEM). Cultures (passage 3; four daysafter plating) grown on LabTek™ permanox slides (Ted Pella, Inc.,Redding, Calif.) were fixed in 2% glutaraldehyde in 100 mM PO₄ at 37° C.for 2 hours and then rinsed and postfixed in 1% aqueous OsO₄ for 1 hourat room temperature. Cultures were then dehydrated in a graded ethanolseries, infiltrated with Araldite resin and polymerized in situ. Theglass slide was separated from the polymerized resin from which blocksof cultured cells were cut and glued to resin blanks. Sections were cutparallel to the culture substrate at a thickness of 70 nm. Sectionscollected on 300 mesh copper grids were stained with uranyl acetate andlead citrate and examined with a Phillips CMIO transmission electronmicroscope at 80 kV.

Scanning Electronic Microscopy (SEM). Cultures.(passage 2; four daysafter plating) grown on LabTek™ glass slides were prepared as for TEM upthrough ethanolic dehydration. The plastic chambers were then removed,leaving the sealing gasket in place, and the slide was placed into aPelco critical point dryer. Following drying, the slide was coated withgold-palladium to a thickness of 300 A in a Technics sputter coater. Thecells were examined in a Cambridge Stereoscan 360 scanning electronmicroscope at 10 kV.

Gene Transfer into Neurons. Approximately 1×10⁶ producer cells wereplated on PORN/laminin coated wells in a 6 well plate and grownovernight at 37° C. in 5% CO₂. Virus from producer cells was collectedafter overnight incubation in DMEM (Dulbecco's minimum essential medium)containing 5% fetal calf serum (FCS) or 5% bovine calf serum (BCS).Virus containing media was filtered through 0.45 μm filters and thenmixed with polybrene (8 μg/ml) and bFGF (20 ng/ml). Media was removedfrom neuronal cultures and virus containing media was added to neuronalcultures and incubated overnight at 37° C. in 5% CO₂. After thisinfection period, the media was removed and replaced with N2 mediacontaining 20 ng/ml bFGF. When the expression vector contained theneomycin resistant gene, the infected cells were selected in thepresence of G418 (400 jig/mi). Cells were passaged and maintained asdescribed above.

The expression vectors and producer cells used were as follows:

1. Avian v-myc gene was expressed from MLV-LTR promoter and bacterialneomycin resistant gene was expressed from thymidine kinase (TK)promoter (Ryder, et al., J. Neurobiol., 21:356-375, 1989; Kaplan, etal., J. Virol., 61:1731-1734, 1987; and Land, et al., Mol. Cell. Biol.,6:1917-1925, 1986). A producer line was generated from ψ2 cells. Thesecells grew in DMEM containing 10% FCS and 400 μg/ml G418. The day beforethe infection, the medium was changed with fresh DMEM containing 5% FCS.

2. Bacterial β-galactosidase gene was expressed from the MLV-LTRpromoter. This expression vector contains a part of the gag gene andproduces very high titer virus. There is no neomycin resistant gene inthis vector. This vector was from Dr. Richard Mulligan, MIT, Cambridge,Mass.

A promoter line was generated from CRIP cells. These cells grow in DMEMcontaining 10% BCS. The day before the infection, the medium was changedwith DMEM containing 5% BCS.

Example 2 Growth of Neurons In Vitro

The chemically defined medium, N2 (Bottenstein and Sato, Proc. Natl.Acad. Sci. USA, 76: 514-517, 1980; Bottenstein, J. E., In: Cell culturein the neurosciences, J. E. Bottenstein and G. H. Sato, Eds., PlenumPress, New York, N.Y., pp 3-43, 1985; di Porizo, et al., Nature,288:370-373, 1980), has been used to reproducibly generate short-termvirtually pure neuronal cultures (Bottenstein, et al., Exp. Cell Res.,125:183-190, 1980; Barnes and Sato, Anal. Biochem., 102:255-302, 1980).This medium does not support the survival or proliferation ofnon-neuronal cells and it is possible to obtain>95% pure neuronalculture. In defined medium, primary cultures of hippocampal neurons diewithin 7 days but can be maintained for 2-4 weeks in the presence ofhippocampal explants, a feeder layer of astrocytes, inastrocyte-conditioned medium (Banker, G. A., Science, 209:809-810, 1980)or in the presence of bFGF (Walicke and Baird, Proc. Natl. Acad. Sci.USA, 83:3012-3016, 1986; Walicke, P A, J. Neurosci., 8:2618-2627, 1988;Walicke, et al., In: Prog. Brain Res., vol. 78, D. m. Gash and J. R.Sladek, Eds. (Elsevier Science Publishers B.V.), pp 333-338, 1988).However, cells continued to die slowly and few cells remained after 1month (Walicke, P., et al., Proc. Natl. Acad. Sci. USA, 83:3012-3016,1986).

bFGF at 20 ng/ml, a concentration of about 100 fold higher concentrationthan that used before to study the survival and elongation of axons(Walicke, P. et al., Proc. Natl. Acad. Sci. USA, 83:3012-3016, 1986.;Walicke, P A, J. Neurosci., 8:2618-2627, 1988), showed dramaticproliferative effects on hippocampal cells. This proliferative propertyof bFGF was used to promote continued proliferation of primaryhippocampal cells to form a long-term culture. Cells cultured in 20ng/ml bFGF began proliferating by 2 days, with a doubling time of 4days. Primary cells became contact inhibited for growth and reached aplateau after day 7, although growth continued within aggregates (FIG.2C).

To test whether division was occurring in ail cells or only in asubpopulation, cultures were incubated with BrdU for 1 or 4 days and thelabeled nuclei were visualized by indirect immunofluorescence using ananti-BrdU-antibody (FIG. 1A, B).

FIG. 1 shows BrdU staining and NeuroTag”* binding of primary neurons inculture. Primary neurons were labeled with BrdU for 1 day (A) and for 4days (B). Only a few cells were stained on day 1, but by day 4 all cellswere stained, indicating that all cells in the culture wereproliferating. The neuronal nature of primary cells was determined bybinding with tetanus toxin (NeuroTag™) (C). Cell bodies and processes ofall cells in culture were stained. Calibration bar=20 μm. After day 1,the nuclei of only a small fraction of cells were immunostained (FIG.1A) but almost the entire cell population was immunostained after 4 daysof incubation with BrdU (FIG. 1B).

To establish long-term cultures, cells were trypsinized and passaged.The passaged cells (up to 6 passages tested) grew as well as theoriginal culture did. Cells were frozen in liquid nitrogen, thawed andcultured again. When cells at different passage numbers were thawed andre-cultured, they grew equally well regardless of the passage number.Freeze-thawed cells showed the same morphology as the cells keptcontinuously in culture.

Other cells derived from neuronal tissue have also been studied fortheir ability to grow and be maintained in N2 media in the presence ofbFGF. Table 1 shows the optimum concentrations of bFGF for culture ofthe various cell lines.

TABLE 1 REGION OF CNS CONCENTRATION OF bFGF (ng/ml)* Hippocampus 20Septum 100 Striatum 20 Cortex 20 Locus Coeruleus 50 VentralMesencephalon 50 Cerebellum 20 Spinal Cord 20 *Optimum concentration ofbFGF used for culture

Example 3 Characterization of Cells

Several independent criteria were used to show that the cells in thecultures were indeed neurons. These included their morphologicalcharacteristics during growth, expression of neuronal markers andultrastructural analysis by transmission and scanning electronmicroscopy.

Cell morphology in culture was similar to that described for short-termcultures of neurons (Banker and Cowan, Brain Res., 126:397-425, 1977;Banker and Cowan, J. Comp. Neurol., 187:469-494, 1979) (FIGS. 2A, B, C).FIG. 2 illustrates photomicrographs showing the morphological changesthat occur during the culture and passaging of primary neurons. A showsprimary cell culture after 4 days of plating in N2+bFGF containednumerous proliferating and process-bearing cells. B shows primary cells4 days in culture after passage (passage 3). Cells were larger andinterconnected by processes that also increased in size. Smallproliferating cells were visible in the culture. C shows cells passaged(passage 3) and kept in culture for ˜14 days in the presence of bFGFformed aggregates and were interconnected by an extensive network ofprocesses forming a lattice-type pattern (Negative magnification 33×).

Cells were immunostained for several different antigenic markers. Cellswere stained with anti-NF (200 KD) antibody (D); with anti-NSE antibody(E) or with anti-GFAP antibody (F). Although all cells stained with antiNF or anti-NSE antibodies, no cell staining was observed with anti-GFAPantibody. (Negative magnification 33× (D,E); 66× (F)).

Cells began to proliferate by day 2 and newborn cells were small andbipolar in shape. Short processes roughly equal in length to cell bodiesstarted to emerge from parent cells. Over the next 2-3 days, 1 or 2 ofthe processes started to grow rapidly and contacted the neighboringcells (FIG. 2A). By day 7, both the cell bodies and the processes hadincreased in size and an extensive interconnecting network of processeshad formed. This morphological progression resembled hippocampaipyramidal neuronal morphologies previously described in vitro (Bankerand Cowan, Brain Res., 126:397-425, 1977; Banker and Cowan, J. Comp.Neurol., 187:469-494, 1979). When cells growing in culture for 1-2 weekswere passaged, more of these cells had processes than did the cellsnewly cultured from the brain (FIG. 2B). It is possible that many ofthese processes survived passaging, albeit partially amputated. Cellspassaged and kept in culture for 14 days in the presence of bFGF formedaggregates and were interconnected by an extensive network of processesforming a lattice-type pattern (FIG. 2C). Few cells divided in openareas; most cell division occurred in the aggregates.

The cultures were characterized by immunostaining for differentantigenic markers (FIGS. 2D, E, F; Table 1). All cell somata and theirprocesses immunostained strongly with an antibody against NF proteinwhich is specifically expressed by neurons (FIG. 2D). Similarly,anti-NSE antibody stained all cells in our culture (FIG. 2E; Table 1).The neuronal nature of the cells proliferating in response to bFGF wasfurther demonstrated by the binding of tetanus toxin, a specific markerfor neurons (Neale, et al., Soc. Neurosci. Abst., 14:547, 1988).NeuroTag™ green stained cell bodies and processes of all cells in theculture (FIG. 1C), indicating that the cells were neurons and that no orvery few non-neuronal cells were present in the cultures. The largeoptical depth of field with the objective used (10×) fails todemonstrate the localization of NeuroTag™ signal as membrane bound.

The cultures were tested by immunostaining for the presence ofnon-neuronal cells (Table 2). Lack of immunostaining with antibodiesagainst GFAP indicated the absence of astrocytes (FIG. 2F). In a controlexperiment anti-GFAP antibody (Amersham), at the same concentration(1:10,000) immunostained rat C6 and 9L and human U373 glioma cells. Theabsence of oligodendrocytes and fibroblasts in our cultures wasdemonstrated by the lack of staining for Gal C, vimentin or fibronectin(Table 2). As a control, rat C6, 9L and human U373 glioma cells werestained with vimentin (1:800) at the same concentration as used forneuronal cultures. The results of immunostaining for other antigenicmarkers are shown in Table 2; these data support the conclusion that thecultures consist of neurons uncontaminated by non-neuronal cells.

TABLE 2 PROPERTIES OF PRIMARY HIPPOCAMPAL NEURONS-ANTIGENIC MARKERS FORNEURONS AND NON-NEURONAL CELLS CULTURING CHARACTERISTICS SubstrateDependency Yes Basic FGF Dependency Yes Freeze-Thaw Viability YesANTIGENIC MARKERS CELL SPECIFICITY Neurofilament Neurons ++^(a) (NF)GFAP Glia −^(b) Nestin Stem cells ++ Vimentin Gliaprecursors/fibroblasts − NSE Neurons +^(c) OX-42 Microglia/macrophages −Galactocerebroside Oligodendrocytes − MAP2 Dendrites + Basic FGFNeurons/glia + receptor Fibronectin Fibroblasts − ++^(a)cells werelabeled strongly −^(b)cells were not labeled +^(c)cells were labeledweakly

Example 4 Analysis of Perpetualized Neurons In Vitro

Analysis of primary neurons in culture at the ultrastructural leveldemonstrated the histotypic neuronal morphology of these cells (FIGS. 3and 4), in agreement with previous ultrastructural studies (Bardett andBanker, J. Neurosci., 4:19440-19453, 1984; Rothman and Cowan, J. Comp.Neurol., 195:141-155, 1981; Peacock, et al., Brain Res., 169:231-246,1979). FIG. 3 shows transmission electron micrographs of primary neuronsin culture. A shows a pyramidal-shaped primary hippocampal neuronshowing both the soma and processes, including a major apical process(arrow) and a finer caliber process (arrowhead). Bar=10 μm. B shows anenlarged view of the neuronal soma shown in panel A. Bar=1 μm. C shows aportion of the major apical process of the neuron shown in panel A. Thisprocess is dominated by microtubules and polysomia! ribosomesidentifying it as a primary dendrite. Bar=1 μm. D shows contact betweentwo neuritic processes. Bar=0.1 μm.

The well-differentiated neurons exhibited a histotypic pyramidalmorphology. including a primary, apical dendrite with multipleramifications, finer caliber axons, and characteristic nuclearmorphology (FIGS. 3 and 4). A TEM micrograph of a pyramidal-shapedprimary hippocampal neuron is shown in FIG. 3A. The level of thissection encompasses both the soma and processes, including a majorapical process (arrow) and a finer caliber process emerging from thebasal aspect of the soma (arrowhead). Other processes from adjacentneurons are also seen. The soma of the neuron has a euchromatic nucleuswith a peripheral rim of heterochromatin and a somewhat reticulatednucleolus (FIG. 3B). Mitochondria and microtubules are present in theperikaryal cytoplasm, which is dominated by rosettes of polysomalribosomes. A portion of the major apical process of the neuron isdominated by microtubules and polysomal ribosomes identifying it as aprimary dendrite (FIG. 3C). Contact between 2 neuritic processes isshown in FIG. 3D. The larger process containing a mitochondrion,microtubules and vesicles is being contacted by a swollen, bouton-likestructure arising from a finer caliber process. The junction betweensuch processes is typically vague and immature at this age in culture.Although the membranes at the site of contact appear to be uniformlyparallel, there is little indication of further assembly of synapticstructures. The contents of the bouton-like process ending are unclear,appearing to be an accumulation of vesicles, with a possible coatedvesicle near the site of contact.

FIG. 4 shows scanning electron micrographs of primary neurons inculture. A shows an overview of primary hippocampal neurons in cultureincluding well-differentiated pyramidal somata (arrow) with largeprocesses containing multiple levels of branching andless-differentiated, rounded neurons with large, extended processes(arrowheads). Bar=50 μm. B shows a major apical dendrite emerging from awell-differentiated pyramidal neuron showing a smooth, regular caliberprocess just proximal to the first (major) bifurcation with severalsmaller processes, possibly axons emerging from it. The PORN/laminincoating the vessel surface can be seen as a porous carpeting which isabsent in some patches. Bar=2 μm. C shows a well-differentiated neuron(in the middle of the field) possessing a large pyramidal soma (compare-to FIG. 3A) and a large apical dendrite (arrowheads) contacted by anumber of processes from other neurons. Other less-differentiatedneurons which are fixed in the process of dividing were also present(arrows). Bar=20 μm. D shows an enlarged view of the dividing neuron inthe upper field of view in panel C. The membrane connecting the twodaughter cell components is clearly continuous, although cytokinesis isapparently underway. Note the process extension from thisless-differentiated neuron, indicating some degree of differentiationduring mitosis. Bar=10 μm.

Scanning EM of primary hippocampal neurons in culture showed thediversity of morphologies present, with some well-differentiatedpyramidal somata (FIG. 4A; arrow) extending large processes which showmultiple levels of branching and some less-differentiated, roundedneurons. Even these rounded neurons possess large, extended processes(FIG. 4A; arrowheads). Closer examination of the major dendriticprocesses arising from the well differentiated neurons shows largecaliber processes with acute bifurcations (FIG. 48). A number of smallcaliber, axon-like processes are seen emerging from these major apicaldendrites (FIG. 48). Well-differentiated neurons typically possess alarge pyramidal soma (FIG. 4C compare to FIG. 3A). Whenless-differentiated neurons are examined, many of these are found tohave been fixed in the process of dividing (FIG. 4C; arrows). A closerview of the dividing neuron shows that although cytokinesis isapparently underway, the membrane connecting the two daughter cellcomponents is clearly continuous. The daughter cell component to theright is extending a fine caliber, possibly axonal, process into theforeground. Extending from this component into the upper right of thefield is another thicker, dendrite-like process which undergoes severallevels of branching.

In contrast to the previous ultrastructural reports (Banker and Cowen,Brain Res. 126:397-425, 1977; Banker and Cowen, J. Comp. Neurol.,187:469-494, 1979; 29 Rothman, et al., J. Comp. Neurol., 135:141-155,1981), the perpetualized neurons had fine caliber axonal processes whichemerged from the soma in a histotypic manner in addition to thedendritic origin (FIGS. 3A and 4D). These somatic axonal extensions maybe the result of the high levels of trophic support. Less-differentiatedneurons typically had rounder somata with fewer, less elaborateprocesses. Even rounded neurons, differentiated adequately to extendprocesses, appeared capable of proliferating (FIG. 40). Neuronalprocesses and somata have been identified based on both theultrastructural surface morphology and organelle content, which clearlydemonstrates that both the well-differentiated and proliferating,less-differentiated cells are neurons.

Example 5 Effects of Different Growth Factors on Cell Culturing

Tissues were dissected from the specific areas of the central nervoussystem (CNS) and dissociated as described in EXAMPLE 1. Aftercentrifugation, cells were resuspended in N2 medium and cells werecounted. Approximately 0.5-1.0×10⁶ cells were plated on PORN/laminincoated 24 well plates in N2 medium containing different growth factorsat different concentrations, depending on the specific region of theCNS. Cells were cultured at 37° C. in 5% CO₂ Cells were examined, and ifnecessary, counted in 5 separate areas in a well at day 1, 4, and 7 todetermine the growth rates in the presence of various growth factors(TABLE 3).

In some experiments, no proliferation of cells was observed in thepresence of certain growth factors. In some cases there was massive celldeath, although a small population of cells survived up to day 4. Thesesurviving cells did not look healthy, however, addition of bFGF at20-100 ng/ml (depending on the origin of the tissue), in N2 mediumrescued these surviving cells as evidenced by this proliferation (see a,TABLE 3).

TABLE 3 EFFECTS OF DIFFERENT GROWTH FACTORS ON PROLIFERATION OF CNSNEURONS Growth Region Factor Concentration Effect Hippocampus bFGF 20ng/ml ++ NGF^(a) 20 ng/ml − EGF 20 ng/ml + BDNF 20 ng/ml − NT3 ND* +Septum bFGF 100 ng/ml  ++ NGF^(a) 100 ng/ml  − EGF^(a) 100 ng/ml  −BDNF^(a) 100 ng/ml  − NT3^(a) ND* − Locus Ceruleus bFGF 50 ng/ml ++ NT3.ND* − Ventral bFGF 50 ng/ml ++ Mecencephalon BDNF 50 ng/ml − EGF 50ng/ml − Cerebellum bFGF 20 ng/ml ++ EGF 20 ng/ml + NGF 20 ng/ml − BDNF50 ng/ml − NT3 50 ng/ml − Spinal Cord bFGF 20 ng/ml ++ NT3 20 ng/ml +*conditioned medium from genetically modified fibroblasts expressing NT3was used; ND—not determined ++ high proliferation − no proliferation +moderate proliferation ^(a)cells could be rescued and proliferated bybFGF

Example 6 Preparation of Adult Neuronal Cultures

Hippocampi of normal adult Fisher rats were dissociated and grown inserum-free culture containing bFGF as described in Example 1. Briefly,hippocampi were dissected from normal adult (>3 mo) rat brains. Most ofthe choroid plexus, ependymal lining and sub-ependymal zone was removed.Cells were dissociated mechanically and enzymatically using methodsdescribed previously (Ray, et al., 1993, supra) with the followingmodifications: After enzymatic dissociation in a papain-protease-DNase(PPD) solution (Hank's balanced salt solution supplemented with 4 mMMgSO₄ and 0.01% papain, 0.1% neutral protease and 0.01% DNasel), cellswere centrifuged at 1000 g for 3 min, resuspended and triturated in 1 mlof 0MEM:F12 (1;1) high glucose medium (Irvine Scientific)+10% fetalbovine serum (10% FBS) (Sigma). Cells were plated onto uncoated plasticT-75 culture flasks (Costar) at 1×10⁶ viable cells per flask in 10% FBSmedium overnight Lower cell densities were used with smaller cultureflasks or Lab-Tek slide chambers (Nunc). Cells were occasionally platedonto cuitureware previously coated with polyomithine/laminin asdescribed in Example 1. The medium was removed the next morning andreplaced with serum-free medium: DMEM:F12+N2 (G1BCO) at 1 ml/100 mlmedium (N2), +bFGF (recombinant human bFGF, Syntex/Synergen Consortium;(Ray, et al., supra) at 20 ng/ml. Flasks were incubated 1-3 weeks, whenhalf of the medium was removed and replaced with the same volume offresh N2+bFGF. Partial medium exchange was made 1-2× weekly or asneeded. Cultures were examined and photographed using phase contrastmicroscopy (Nikon Diaphot).

In a number of experiments cells were harvested and transferred directlyto new flasks or Lab-Tek slide chambers where they attached immediatelyand started proliferating, or occasionally passaged using trypsinizationwith ATV trypsin (Irvine Scientific), followed by washing,centrifugation and re-plating in N2+bFGF.

Primary cultures of neurons from adult rat hippocampi were replicatedmore than 15 times. To determine whether 10% FBS or N2 medium couldaccount for the observed effects, some cultures were grown in 10% FBS orN2. Only cultures with bFGF developed large numbers of neurons. Somedissections were made of the CA1, CA3 and dentate gyrus regions. Neuronswere generated from all three regions. Cultures are described in threeoverlapping temporal stages: early, middle and late.

Early cultures (1-21 days) were characterized by cell attachment to thesubstrate, cell proliferation and expression of mature neuronalfeatures. After clearing cell debris in the medium, single cells thatwere phase-bright and round and doublet cells, suggestive of celldivision, were observed at two days in vitro (d.i.v.). Numerousphase-bright cell bodies displayed processes tipped with growth cones.Cells of neuronal morphologies, i.e., phase-bright multipolar cell bodywith thin branching processes, were observed as early as 5 d.i.v.Processes developing complex branching patterns and evidence ofincomplete cytokinesis or potential synapse formation betweenpresumptive sibling neurons were observed as earty as 8 d.i.v. (Nikonphase contrast-2 microscope/negative magnification 33×-66×).

For SEM, cultures were fixed in 2% glutaraldehyde in 0.1 M PBS,osmicated in 1% aqueous osmium tetroxide, dehydrated in a graded ethanolseries, critical point dried with liquid carbon dioxide, attached tostubs with silver paste, sputter coated to 300 A with gold/pailadium andexamined and photographed in a Cambridge Scanning Bectron Microscope(Stereoscan 360).

Examination of the three-dimensional morphology of earty/intermediatestage cultures using scanning electron microscopy (SEM) revealednumerous cells of both neuronal and epithelioid phenotypes. Lacy neuralnetworks were observed as with phase microscopy. Cells that appeared tobe dividing were also observed. Higher magnification revealed that theprocesses between cells and cell aggregates interpreted at the lightmicroscope level as a single process were frequently 2 or morefasciculated processes.

Intermediate cultures (approximately 14-60 days) were characterized byincreasing numbers of cells, the presence of neural networks, thedevelopment of mature neurons and initial cell aggregate formation.Rudimentary networks of fine processes connecting small cell clusterswere observed as early as 14 d.i.v. Networks of cells displayingneuronal morphologies became more extensive and complex. Cells in thecultures were heterogeneous although individual patches of neuralnetworks displayed a uniform morphological phenotype. Individual cellsaway from clusters or networks also developed well differentiatedmorphological features characteristic of mature neurons, with largephase-bright multipolar cell bodies and long thin processes thatbranched repeatedly. Processes of these cells often measured nearly 1000um, and large indented nuclei and prominent nucleoli could be seen indifferent focal planes. Some neurons displayed small thorn-likeprojections indistinguishable from dendritic spines on processes.

Late cultures (approximately 2 to 7 months) were characterized byincreasing numbers of cells to confluence, increasing cell aggregatesconnected by processes and a background of individual cells. Whensubstrate space was available, cells with multiple thin processescharacteristic of earlier stages continued to be observed. Cellaggregates were connected by cable-like neurites. Large numbers of cellaggregates developed and the entire substrate became covered with cellaggregates and individual cells that appeared to have migrated from thecell aggregates. While many background cells displayed features typicalof neurons, some cells expressed features typical of astrocytic gfia.

Example 7 Gene Expression in Cultured Neuronal Cells

The presence of NFh and GFAP was further confirmed by reversetranscriptase-polymerase chain reaction (RT-PCR) with RNA obtained fromcells harvested after different times in culture.

RNA was extracted using the guanidinium-cesium chloride (CsCl) method(Current Protocols in Molecular Biology, Vol. 1, Wiley Intersciencs, NY,F. M. Ausubel, et al., eds, 1988). The pellets were solubilized in 1 mlsolution D (4.0M guinidine thiocyanate, 25 mM Na citrate, 0.5% sarcosyland DEPC treated H₂O) after thawing, triturated gently and the celllysate was transferred to CsCl previously poured into centrifuge tubes.The level of the CsCl was marked, and the tubes were weighed aridbalanced. The tubes were centrifuged in a Beckman Ultracentrifugeovernight at 40,000 rpm at 20° C. The next morning, solution D wasremoved, and the interface washed with solution D. The CsCl solution wascarefully poured off, and the RNA pellet was rinsed with 70% EtOH (madewith DEPC water). After the pellet was dry it was solubilized in DEPCH₂Oand the remainder was stored in EtOH at −70° C.

A RT-PCR method was used to obtain cDNA's (Ausubel, et al., 1988,supra). The reaction tube contained 4 μl RNA (10-100 ng). 8 μl(sufficient DEPC—H₂O to bring the volume up to 20 μl), 2 μl 10× PCRbuffer, 2 μl 10 mM d NTP's, 1 μl random hexamers, 3 μl 24 mM MgCl, 0.125μl AMV-RT and 0.5 μl RNasin. A drop of Nujol mineral oil was added toeach tube and the reaction was run in a Perkin Elmer Thermal Cyder 42°C.-75 min; 95° C.-10 min; and held at 4° C.

A PCR method was used to further amplify the specific desired cDNAs fromthe cDNAs obtained above. Each reaction tube contained 5 μl cDNA, 9.5 μlPCR buffer, 7.25 μl MgCl₂, 0.2 or 0.3 μl ³²P-dCTP, 1.5 μl 10 mM dNTP's,0.5 Taq polymerase, 6μ, 1 primers (2 μl [1 μl (F (Forward rx):5′)+1/μl R(reverse:3′) each of RPL 27, NFh, GFAP, NGF or bFGF) and sufficient H₂Oto bring the volume to 100 μl. The reaction was run in a Perkin ElmerThermal Cycler: 94° C.-10 min and held at 4° C.

AmpliTaq DNA polymerase was from Perkin-Elmer, AMV reversetranscriptase, random oligonucleotide hexamer primers and recombinantRNasin ribonuclease inhibitor were from Promega, specific primers weremade to order. dNTP's were from New England Nuclear. The primers were asfollows:

NFh Forward (F) primer: 5′-GAGGAGATAACTGAGTACCG-3′ (SEQ ID NO: 1)Reverse (R) primer: 5′-CCAAAGCCAATCCGACACTC-3′ (SEQ ID NO: 2) GFAP Fprimer: 5′-ACCTCGGCACCCTGAGGCAG-3′ (SEQ ID NO: 3) R primer:5′-CCAGCGACTCAACCTTCCTC-3′ (SEQ ID NO: 4)

Gel electrophoresis of cDNA-samples obtained from PCR amplification wasdone on a 6% non-denaturing polyacrylamide gel. Some samples and theircorresponding digests were run on agarose gels using ethidium bromide tobind and illuminate the DNA under UV light A 123 bp molecular ladder wasrun in a lane beside the samples. Electrophoresis was done for varyingperiods of time, and the resulting gels were dried for 1 hr on a geldrier. Autoradiographic films of dried acrylamide gels were developedfor periods of time ranging from several hours to 10 days.

Relative levels of mRNA were analyzed quantitatively using densitometryover cDNA bands identified as NFh, GFAP, NGF and bFGF from Northernblots of cultures grown 36 to 117 days. A diverging pattern of mRNAexpression was apparent. Expression of message for NFn was relativelylow. At about 2 months, the relative levels switched and expression ofmRNA for GFAP increased over time then dropped dramatically at about 4months in culture, while expression of NFh fell over time, then roseslightly after 4 months in culture.

Digests of NFh were performed on samples remaining from earlierreactions using a cocktail consisting of 40 μl sample, 5 μl React #6buffer (50 mM Tris, pH 7.4, 6 mm MgCl₂, 50 mM KCl, 50 mM NaCl) and 5 μlPvu II restriction enzyme, and reacted for 1 hr at 37° C. The productswere run along with a 123 bp molecular ladder on a 6% acrylamide gel.The gel was dried, exposed on film for varying periods of time, and theresulting autoradiograms were examined for bands at the predictedmolecular weight levels. mRNA for both NFh and GFAP was present in ailcultures at the times examined.

Example 8 Immunocytochemistry

To determine whether cells expressed antigens typical of neural tissue,cultures were processed for immunocytochemistry. Cells were fixed for 30min in 4% paraformaldehyde at room temperature or 37° C., incubated with0.6% H₂O₂ in TBS followed by incubation in blocking solution. Thecultures were incubated with primary antibody at appropriate dilutionsovernight at 4° C. The next day cells were rinsed with diluent andincubated in secondary antibody for 1 hr at room temperature, rinsedwith TBS and incubated in ABC solution (equal amounts of avidin andbiotin) for 1 hr at room temperature. They were rinsed with TBS andincubated with DAB-NiCl for variable reaction times, rinsed with TBS,dried overnight dehydrated through graded series of alcohol and mountedin histoclear. Antibodies were from the following sources and used atthe dilutions indicated. Monoclonal antibodies: high molecular weightsub-unit of neurofilament protein (NF-H 200 kD; 1:24); middle molecularweight sub-unit of neurofilament protein (NF-M 160:1:10); glial filamentacidic protein (GFAP; 1:100) and synaptophysin (1:10) (BoehringerMannheim); calbindin (Cal-b; 1:200) and microtubule associated protein 2(MAP2; 1:500) (Sigma); neuron-specific enolase (NSE; 1:200) (DAKO).Polyclonal antibodies: NF-H 200 (1:250); NF-M 150 (1:500); NF-L 68(1:125); GFAP (1:1000) and gamma amino butyric acid (GABA; 1:200)(Chemicon); NSE (1:800) (Polysciences); galactocerebrocide (Gal-C;1:5000) (Advanced Immuno Chem.); bFGF (1:1000); (Whittier Institute, LaJolla, Calif.). Normal horse and goat serum, biotinylated goatanti-rabbit IgG, horse anti-mouse IgG and ABC Vectastain Elite kit werefrom Vector Laboratories (Burlingame, Calif.). There was no detectablestaining when primary antibody was omitted and replaced with non-immuneserum.

Neuro-specific enolase (NSE)-positive cells-were observed in, earlycultures. By 170 d.i.v., a majority of the cells were immunoreactive forhigh molecular weight neurofilament protein (NFn, 200 kD) that ischaracteristic for adult neurons, as well as the middle and lowmolecular weight of NF. Most NF-positive cells also had morphologicalproperties of neurons. A small subpopulation of cells (less than 10%)was immunopositive for calbindin, which is specific for granule cells.Cells with neuronal phenotypes were also immunopositive for MAP2 withreaction product localized to the cytoplasm of cell bodies and proximalprocesses. Cells with astroglial morphology stained for GFAP. Less than1% of the cells stained for GABA. Many cells were immunopositrve forbFGF and a few small round cells were immunopositive forgalactocerebroside.

Example 9 Characterization of Neuronal Cell Growth In Vitro

To determine whether cells were proliferating and, if so, assess thenature of such cell types, cultures were incubated in bromodeoxyuridine(BrdU) for 36 hours and then dual labeled for immunofluorescence withBrdU and neuron-specific enolase (NSE) or glial filament acidic protein(GFAP).

For BrdU incorporation, cells were cultured in glass Lab-Tek slidechambers for 11 days. The medium was replaced with fresh N2+bFGFcontaining bromodeoxyuridine (BrdU) labeling reagent (1 μl/ml medium;Amersham) and the cultures were incubated an additional 1 or 4 days fora total of 12 or 15 days. Cells were fixed in 4% paraformaldehyde inphosphate-buffered saline (PBS) for 30 min, washed with PBS, blockedwith 10% normal donkey serum (Jackson ImmunoResearch Labs) in PBS, andreacted with monoclonal antibody to BrdU (BrdU; undiluted; Amersham)followed by donkey anti-mouse IgG coupled to Cy-5 (JacksonImmunoResearch Labs). Some cultures were dual labeled with polyclonalantibody against neuron specific enolase (NSE; 1;800) or antibodyagainst glial fibrillary acidic protein (GFAP; 1:1000). Secondaryantibody for the polyclonals was donkey anti-rabbit IgG conjugated tofluorescein isothiocyanate (FITC; Jackson ImmunoResearch Labs). Slideswere mounted in Slow-Fade mounting reagent (Molecular Probes). Cellswere visualized using a BioRad MRC 600 Confocal Scanning LaserMicroscope. Images were collected and transferred to an Apple MacintoshQuadra 700, merged using Adobe Photoshop 2.01 and printed out on a GCCfilm recorder.

Confocal scanning microscopy revealed cells immunoreactive for NSE andBrdU, as well as BrdU and GFAP positive cells showing that cellsexpressing neuronal and glial cell markers dividing in these cultures.

To determine if cell numbers in culture were increasing, cells werecounted over a 2 week period in 10 random fields. Thirty-seven percentof cells originally attached had survived by the second day in culture.Within 5 days, cell numbers had risen to slightly above their originallevel, and by the end of seven days, there were nearly twice as manycells. By the end of 2 weeks, there were almost five times as many cellsas on the first day in culture.

The most important result of this study is the demonstration of neuronalproliferation from normal adult hippocampus when cultured with bFGF.Neurons survive and proliferate abundantly for long periods of time,more tharf 200 d.i.v. to date; this is the first such demonstration.

It has been reported that initiation of cell division of isolated adultbrain (striatal) cells in culture requires epidermal growth factor(EGF), but not bFGF at 20 ng/ml, and a non-adhesive substrate (Reynolds& Weiss, Science, 755:1707, 1992). In contrast, the present datasupports that: 1) bFGF at 20 ng/ml acts as a strong mitogen and as asurvival factor in adult as well as fetal hippocampal cultures; 2)proliferation occurs in substrate-bound cells and aggregates, i.e.,cells not in suspension, and 3) many, if not most, of the cells thatattach are bFGF-responsive.

Limited neuronal division as been reported over short times in otherculture systems of adult bran (Reynolds, supra; Richards, et al. Proc.Natl. Acad. Sci., USA, 89:8591, 1992). In the present studyproliferation was confirmed by BrdU incorporation and the finding thatcell numbers increased almost 500% over a 2 week period. Although it hasbeen reported that less that 1% of adult striatal cells initially platedproliferate (Reynolds, et al., supra), in the cultures described hereinnearly 40% of cells which initially attached survived to the second dayin culture, suggesting that many cells are present in adult hippocampusthat have the capacity to proliferate.

Evidence from several independent experiments supports the idea thatmost cells in these cultures not only are neurons, but they are matureneurons which express morphological, biochemical and molecular featurescharacteristic of adult neurons. While glia are also generated, gliawere a minority phenotype in most cultures. Similar findings have beenreported for fetal rat hippocampus neurons cultured with bFGF (Ray, etal., 1993, supra).

The source of the proliferating neurons for the adult brain remains tobe determined. The cells could be mature functioning neurons that weresaved and induced to proliferated by high concentration of bFGF; thecells could be stem cells of suspected proliferation zones; or the cellscould be partially committed neurons (neuroblasts) that have becomequiescent due to a reduction in high levels of bFGF present only in theembryonic brain and/or because of contact inhibition. While it isunlikely that all neurons that were observed and generated could beaccounted for the mature neurons saved following plating, work is inprogress to determine whether mature differentiated neurons are capableof in vitro survival and proliferation through dedifferentiation. It ispossible that the subventricular zone (SVZ) could be the source of thesecells, since SVZ of mammalian forebrain has been shown to be the sourceof these cells that differentiate into neurons and glia in adult mice(Clois, et al., Proc. Natl. Acad. Sci. U.S.A., 79:2074, 1993). However,it is not likely that the SVZ could have served as the main source ofproliferating cells in the cultures of the invention, since theependymal lining/SVZ, along with choroid plexus, was stripped away.These neurons could be derived from a small population of embryonic stemcells that survives in the adult brain in a dormant, non-proliferativestate, as has been suggested exists in adult mouse striatum (Reynolds,et al., supra). Alternatively, these neurons could be neuronal precursorcells existing in adult mammalian brain that require discrete epigeneticsignals for their proliferation and differentiation as has beenspeculated for adult mouse brain (Richards, et al., supra).

In addition to stem cells of the SVZ, there is a large population ofneuroblast in the normal adult mammalian hippocampus that can be inducedto generate large numbers of neurons over long periods of time underappropriate in vitro conditions. It is possible that this could also betrue in vivo, a concept that has profound implications for basic andclinical neuroscience. This could mean that normal hippocampus and, byextension, normal CNS has a reservoir of cells that can be activatedunder appropriate conditions to replicate large numbers of neurons.Thus, neuroblasts could be present not only in cultures of fetal CNS,but also in cultures of adult CNS and in the adult CNS in situ.

Example 10 Long-Term Culture of Neurons From Adult Hippocampus

Brains of adult Fisher rats (>3 months old) were dissected, themenengies removed, and the hippocampal dissected out The tissues weretransferred to a 15 ml tissue culture tube and washed three times with 5ml Dulbecco's phosphate buffered saline (D-PBS). After the last wash,the tissue was pelleted by centrifugation at 1000 g for 3 min and thewash solution removed. The tissue was suspended in 5 ml papain-neutralprotease-DNase (PPD) solution and incubated at 37° C. for 20-30 min withoccasional shaking. The solution was made in Hank's balanced saltsolution supplemented with 12.4 mM MgSO₄ containing 0.01% papain, 0.1%neutral protease and 0.01% DNase I (London, R. M. and Robbins, R. J.,Method. Enzymol., 124:412-424, 1986).

Hippocampal were mechanically dissociated by tituration with a mediumbore pasteur pipet (about 20 times). Cells were pelleted bycentrifugation at 1000 g for 3 min. The cells were resuspended in 1 mlDMEM:F12 (1:1) medium containing 10% fetal bovine serum, 3.9 mMglutamine (complete medium). Cell clumps were mechanically dissociatedby tituration with medium to fine bore pasteur pipets (about 20 timeswith each). Cells were washed with 5-10 ml complete medium twice bycentrifugation. Cells were taken up in 1 ml complete medium, dissociatedby tituration and counted in a hemocytometer. Cells were plated at adensity of 1×10⁶ cells/T75 flasks (Coaster) and incubated at 37° C. in5% CO₂ 95% air incubator. After incubation for 18-24 hours, the mediumwas changed with N2 medium [1:1 mixture of DMEM/F-12 containing 20 nMprogesterone, 30 nM sodium selenite, 100 μM putrescine, 3.9 mMglutamine, insulin (5 μg/ml) and transferrin (100 μg/ml)] containing 20ng/ml FGF-2 (bFGF). To date cells have been cultured for at least 7months and have been cultured from 15 different independent dissections.

The neuronal nature of cells were determined by examination ofmorphology at light and scanning microscope levels. Immunocytochemicalanalysis showed that these cells expressed neuron-specific enolase,neurofilament medium and high molecular weight proteins, MAP-2, andcalbindin (only a small population). Some cells in these cultures alsostained for GFAP indicating the presence of astrocytes in thesecultures. The proliferation of adult neuronal cells in cultures wasdetermined by bromodeoxyuridine (BrdU) incorporation. The nuclei ofcells expressing neuron-specific enolase were immunostained with anantibody against BrdU indicating cell proliferation in culture.

Example 11 In Vivo Survival of Perpetual Hippocampal Neurons AfterGrafting in the Adult Brain

Embryonic hippocampal neurons were cultured in N2 medium containing 20ng/ml bFGF. Cells were passaged and allowed to grow until 70-80%confluent. The medium was replaced with fresh medium (N2+bFGF)containing ³H-thymidine (1 μCi/ml; specific activity: 25 Ci/mmol) andallowed to grow for 3.5 days. Cells were harvested from flasks bytrypsinization and washed with D-PBS 3 times by centrifugation. Cellswere resuspended in 2 mls of D-PBS containing 20 ng/ml bFGF, dissociatedby tituration and counted in a hemocytometer. After centrifugation toremove the supernatant, cells were resuspended at a concentration of60,000 cells/μl. One microliter of cell suspension was injected in thehippocampus of adult Fisher rats (>3 months old). Animals were perfusedwith 4% paraformaldehyde, and the brains removed. Brain sections weretreated with antibodies with calbindin, GFAP and NF-H proteins. Thesections were dipped in emulsion and developed after 6 weeks. Number ofcells with at least 12 grains on them were counted in every 12 sectionsfor each animal (3 animal total). At three weeks, an average of 17%cells implanted in the brain survived (Table 4).

TABLE 4 SURVIVAL OF PERPETUAL HIPPOCAMPAL NEURONS IN ADULT RATHIPPOCAMPUS AVERAGE # CELLS % CELLS ANIMAL WITH GRAINS/SECTION SURVIVING1 222 15 2 233 19 3 244 17

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention.

1. A composition comprising: a population of isolated neural cellscomprising a neuroblast and a nestin-positive neural cell, wherein theisolated neural cells are derived from the CNS of an adult mammal, and aculture medium comprising about 1 ng/ml to 100 ng/ml of basic fibroblastgrowth factor (bFGF); wherein the population has been continuouslyproliferating in vitro greater than 21 days, and wherein the compositionis free of epidermal growth factor (EGF).
 2. The composition of claim 1,wherein the doubling time of the population of isolated neural cells isless than 10 days.
 3. The composition of claim 1, wherein the populationof isolated neural cells is capable of giving rise to glial cells andneurons.
 4. The composition of claim 1, wherein the isolated neuralcells are not genetically modified.
 5. The composition of claim 1,wherein the culture medium further comprises leukemia inhibitory factor(LIF).
 6. The composition of claim 1, wherein the nestin positive neuralcell is an undifferentiated cell capable of giving rise to a glial cellor a neuron.
 7. The composition of claim 1, wherein the isolated neuralcells are derived from the adult mammalian hippocampus.
 8. Thecomposition of claim 7, wherein the isolated neural cells are derivedfrom the dentate gyrus region of the adult mammalian hippocampus.
 9. Thecomposition of claim 1, wherein the isolated neural cells arepredominantly of neuronal lineage.
 10. The composition of claim 1,wherein the isolated neural cells are predominantly neurons.
 11. Thecomposition of claim 1, wherein the adult mammal is a human.
 12. Thecomposition of claim 1, further comprising at least one pharmaceuticallyacceptable excipient suitable for transplantation into the CNS of ahuman subject.
 13. The composition of claim 1, wherein the quintuplingtime of the population is about 14 days.
 14. The composition of claim 1,wherein the population has been continuously proliferating in vitrogreater than 60 days.
 15. The composition of claim 1, wherein thepopulation has been continuously proliferating in vitro greater than 200days.
 16. The composition of claim 1, wherein the population furthercomprises a cell immunopositive for calbindin.